dNLS Preprocessing QC statistics ¶

October 2024¶

In [2]:
import os
NOVA_HOME = ''
NOVA_DATA_HOME = ''
LOGS_PATH = os.path.join(NOVA_DATA_HOME, "outputs/preprocessing/spd/logs/dNLS")
PLOT_PATH = os.path.join(NOVA_HOME, 'src', 'preprocessing', 'notebooks','figures','dNLS')
os.chdir(NOVA_HOME)
import pandas as pd
import numpy as np
# plt.rcParams["image.cmap"] = "Set1"
import contextlib
import io
from IPython.display import display, Javascript

from tools.preprocessing_tools.qc_reports.qc_utils import log_files_qc, run_validate_folder_structure, display_diff, sample_and_calc_variance, \
                                                show_site_survival_dapi_brenner, show_site_survival_dapi_cellpose, \
                                                show_site_survival_dapi_tiling, show_site_survival_target_brenner, \
                                                calc_total_sums, plot_filtering_heatmap, show_total_sum_tables, \
                                                plot_cell_count, plot_catplot, plot_hm_combine_batches, plot_hm, \
                                                run_calc_hist_new
                                                
from tools.preprocessing_tools.qc_reports.qc_config import dnls_panels, dnls_markers, dnls_marker_info, dnls_cell_lines, \
                                                dnls_cell_lines_to_cond, dnls_cell_lines_for_disp, reps, \
                                                dnls_line_colors, dnls_lines_order, dnls_custom_palette, \
                                                dnls_expected_dapi_raw, markers, custom_palette

%load_ext autoreload
%autoreload 2
In [4]:
# choose batches
batches = [f'batch{i}' for i in range(3,6)]
batches
Out[4]:
['batch3', 'batch4', 'batch5']
In [5]:
df = log_files_qc(LOGS_PATH, only_wt_cond=False, batches=batches)
df_dapi = df[df.marker=='DAPI']
df_target = df[df.marker!='DAPI']
# we need to match between the raw marker name (TDP43) and the processed marker name (TDP43N / TDP43B)
df_target.loc[df_target['marker'] == 'TDP43', 'marker'] += df_target['panel'].str.replace('panel', '')

reading logs of batch4
reading logs of batch3
reading logs of batch5

Total of 3 files were read.
Before dup handeling  (74261, 22)
After duplication removal #1: (74261, 23)
After duplication removal #2: (74261, 23)

Actual Files Validation¶

Raw Files Validation¶

  1. How many site tiff files do we have in each folder?
  2. Are all existing files valid? (tif, at least 2049kB, not corrupetd)
In [11]:
root_directory_raw = os.path.join(NOVA_DATA_HOME, 'input', 'images', 'raw', 'SpinningDisk','deltaNLS_sort')

raws = run_validate_folder_structure(root_directory_raw, False, dnls_panels, dnls_markers.copy(),PLOT_PATH, dnls_marker_info,
                                    dnls_cell_lines_to_cond, reps, dnls_cell_lines_for_disp, dnls_expected_dapi_raw,
                                     batches=batches, fig_width=5)

batch3
Folder structure is invalid. Missing 2 paths:
/home/labs/hornsteinlab/Collaboration/MOmaps/input/images/raw/SpinningDisk/deltaNLS_sort/batch3/WT/panelN
/home/labs/hornsteinlab/Collaboration/MOmaps/input/images/raw/SpinningDisk/deltaNLS_sort/batch3/TDP43/panelN
No bad files are found.
Total Sites:  25800

========
batch4
Folder structure is invalid. Missing 2 paths:
/home/labs/hornsteinlab/Collaboration/MOmaps/input/images/raw/SpinningDisk/deltaNLS_sort/batch4/WT/panelN
/home/labs/hornsteinlab/Collaboration/MOmaps/input/images/raw/SpinningDisk/deltaNLS_sort/batch4/TDP43/panelN
No bad files are found.
Total Sites:  25500

========
batch5
Folder structure is valid.
No bad files are found.
Total Sites:  26897

========
====================

Processed Files Validation¶

  1. How many site npy files do we have in each folder? -> How many sites survived the pre-processing?
  2. Are all existing files valid? (at least 100kB, npy not corrupted)
In [12]:
root_directory_proc = os.path.join(NOVA_DATA_HOME, 'input', 'images', 'processed', 'spd2',
                              'SpinningDisk','deltaNLS')
procs = run_validate_folder_structure(root_directory_proc, True, dnls_panels, dnls_markers,PLOT_PATH,dnls_marker_info,
                                    dnls_cell_lines_to_cond, reps, dnls_cell_lines_for_disp, dnls_expected_dapi_raw,
                                     batches=batches, fig_width=5)

batch3
Folder structure is invalid. Missing 3 paths:
/home/labs/hornsteinlab/Collaboration/MOmaps/input/images/processed/spd2/SpinningDisk/deltaNLS/batch3/WT/Untreated/TDP43N
/home/labs/hornsteinlab/Collaboration/MOmaps/input/images/processed/spd2/SpinningDisk/deltaNLS/batch3/TDP43/dox/TDP43N
/home/labs/hornsteinlab/Collaboration/MOmaps/input/images/processed/spd2/SpinningDisk/deltaNLS/batch3/TDP43/Untreated/TDP43N
No bad files are found.
Total Sites:  24336

========
batch4
Folder structure is invalid. Missing 3 paths:
/home/labs/hornsteinlab/Collaboration/MOmaps/input/images/processed/spd2/SpinningDisk/deltaNLS/batch4/WT/Untreated/TDP43N
/home/labs/hornsteinlab/Collaboration/MOmaps/input/images/processed/spd2/SpinningDisk/deltaNLS/batch4/TDP43/dox/TDP43N
/home/labs/hornsteinlab/Collaboration/MOmaps/input/images/processed/spd2/SpinningDisk/deltaNLS/batch4/TDP43/Untreated/TDP43N
No bad files are found.
Total Sites:  23638

========
batch5
Folder structure is valid.
No bad files are found.
Total Sites:  25527

========
====================

Difference between Raw and Processed¶

In [13]:
display_diff(batches, raws, procs, PLOT_PATH, fig_width=5)

batch3

========
batch4

========
batch5

========

Variance in each batch (of processed files)¶

In [14]:
for batch in batches:
    with contextlib.redirect_stdout(io.StringIO()):
        var = sample_and_calc_variance(root_directory_proc, batch, 
                                       sample_size_per_markers=200, cond_count=2, rep_count=len(reps), 
                                       num_markers=len(dnls_markers))
    print(f'{batch} var: ',var)

batch3 var:  0.010357440534791738
batch4 var:  0.010663615289945838
batch5 var:  0.01020901013294272

Preprocessing Filtering qc¶

By order of filtering

1. % site survival after Brenner on DAPI channel¶

Percentage out of the total sites

In [15]:
dapi_filter_by_brenner = show_site_survival_dapi_brenner(df_dapi,batches, dnls_line_colors, dnls_panels, reps)

2. % Site survival after Cellpose¶

Percentage out of the sites that passed the previous filter. In parenthesis are absolute values.

A site will be filtered out if Cellpose found 0 cells in it.

In [16]:
dapi_filter_by_cellpose = show_site_survival_dapi_cellpose(df_dapi, batches, dapi_filter_by_brenner, dnls_line_colors, dnls_panels, reps)

3. % Site survival by tiling¶

Percentage out of the sites that passed the previous filter. In parenthesis are absolute values.

A site will be filtered out if after tiling, no tile is containing at least one whole cell that Cellpose detected.

In [17]:
dapi_filter_by_tiling=show_site_survival_dapi_tiling(df_dapi, batches, dapi_filter_by_cellpose, dnls_line_colors, dnls_panels, reps)

4. % Site survival after Brenner on target channel¶

Percentage out of the sites that passed the previous filter. In parenthesis are absolute values (if different than the percentages).

In [18]:
show_site_survival_target_brenner(df_dapi, df_target, dapi_filter_by_tiling, dnls_markers)

Statistics About the Processed Files¶

In [20]:
names = ['Total number of tiles', 'Total number of whole cells']
stats = ['n_valid_tiles','site_whole_cells_counts_sum','site_cell_count','site_cell_count_sum']
total_sum = calc_total_sums(df_target, df_dapi, stats, dnls_markers)

Total tiles¶

In [31]:
markers_for_dnls = markers.copy()
markers_for_dnls.remove('TIA1')
markers_for_dnls += ['TDP43B']
total_sum[total_sum.marker.isin(markers_for_dnls)].n_valid_tiles.sum()
Out[31]:
374956

Total whole nuclei in tiles¶

In [10]:
total_sum[total_sum.marker =='DAPI'].site_whole_cells_counts_sum.sum()
Out[10]:
152391.0

Total nuclei in sites¶

In [12]:
total_sum[total_sum.marker =='DAPI'].site_cell_count.sum()
Out[12]:
385860.0
In [25]:
show_total_sum_tables(total_sum)
n_valid_tiles % valid tiles site_whole_cells_counts_sum site_cell_count
batch3
count 258.000000 258.000000 258.000000 258.000000
mean 584.647287 5.846473 653.767442 1637.193798
std 210.678623 2.106786 245.053556 716.126394
min 123.000000 1.230000 131.000000 285.000000
25% 332.000000 3.320000 356.500000 746.750000
50% 677.500000 6.775000 774.000000 1944.000000
75% 760.000000 7.600000 857.000000 2248.000000
max 891.000000 8.910000 1006.000000 2647.000000
sum 150839.000000 NaN 168672.000000 422396.000000
expected_count 450.000000 450.000000 450.000000 450.000000
n_valid_tiles % valid tiles site_whole_cells_counts_sum site_cell_count
batch4
count 255.000000 255.000000 255.000000 255.000000
mean 543.862745 5.438627 603.854902 1547.223529
std 196.541216 1.965412 225.328069 674.661059
min 40.000000 0.400000 42.000000 81.000000
25% 357.000000 3.570000 399.000000 820.000000
50% 576.000000 5.760000 651.000000 1763.000000
75% 721.000000 7.210000 810.000000 2116.000000
max 887.000000 8.870000 993.000000 2613.000000
sum 138685.000000 NaN 153983.000000 394542.000000
expected_count 450.000000 450.000000 450.000000 450.000000
n_valid_tiles % valid tiles site_whole_cells_counts_sum site_cell_count
batch5
count 270.000000 270.000000 270.00000 270.000000
mean 557.933333 5.579333 622.92963 1564.825926
std 189.977878 1.899779 220.40687 658.346156
min 105.000000 1.050000 114.00000 249.000000
25% 366.750000 3.667500 396.00000 819.750000
50% 603.000000 6.030000 687.00000 1756.500000
75% 731.500000 7.315000 828.50000 2142.000000
max 892.000000 8.920000 1013.00000 2540.000000
sum 150642.000000 NaN 168191.00000 422503.000000
expected_count 450.000000 450.000000 450.00000 450.000000
n valid tiles % valid tiles site_whole_cells_counts_sum site_cell_count
All batches
count 783.000000 783.000000 783.000000 7.830000e+02
mean 562.153257 5.621533 626.878672 1.582939e+03
std 199.577538 1.995775 230.985788 6.833561e+02
min 40.000000 0.400000 42.000000 8.100000e+01
25% 353.000000 3.530000 377.000000 7.900000e+02
50% 619.000000 6.190000 701.000000 1.819000e+03
75% 733.500000 7.335000 825.000000 2.159000e+03
max 892.000000 8.920000 1013.000000 2.647000e+03
sum 440166.000000 NaN 490846.000000 1.239441e+06
expected_count 450.000000 450.000000 450.000000 4.500000e+02

Show Total Tile Counts¶

For each batch, cell line, replicate and markerTotal number of tiles

In [26]:
to_heatmap = total_sum.rename(columns={'n_valid_tiles':'index'})
plot_filtering_heatmap(to_heatmap, extra_index='marker', vmin=None, vmax=None,
                      xlabel = 'Total number of tiles', show_sum=True, figsize=(6,8))

/home/labs/hornsteinlab/Collaboration/MOmaps_Noam/MOmaps/tools/preprocessing_tools/qc_utils.py:397: UserWarning: set_ticklabels() should only be used with a fixed number of ticks, i.e. after set_ticks() or using a FixedLocator.
  for batch in batches:
/home/labs/hornsteinlab/Collaboration/MOmaps_Noam/MOmaps/tools/preprocessing_tools/qc_utils.py:397: UserWarning: set_ticklabels() should only be used with a fixed number of ticks, i.e. after set_ticks() or using a FixedLocator.
  for batch in batches:

/home/labs/hornsteinlab/Collaboration/MOmaps_Noam/MOmaps/tools/preprocessing_tools/qc_utils.py:397: UserWarning: set_ticklabels() should only be used with a fixed number of ticks, i.e. after set_ticks() or using a FixedLocator.
  for batch in batches:
/home/labs/hornsteinlab/Collaboration/MOmaps_Noam/MOmaps/tools/preprocessing_tools/qc_utils.py:397: UserWarning: set_ticklabels() should only be used with a fixed number of ticks, i.e. after set_ticks() or using a FixedLocator.
  for batch in batches:

/home/labs/hornsteinlab/Collaboration/MOmaps_Noam/MOmaps/tools/preprocessing_tools/qc_utils.py:397: UserWarning: set_ticklabels() should only be used with a fixed number of ticks, i.e. after set_ticks() or using a FixedLocator.
  for batch in batches:
/home/labs/hornsteinlab/Collaboration/MOmaps_Noam/MOmaps/tools/preprocessing_tools/qc_utils.py:397: UserWarning: set_ticklabels() should only be used with a fixed number of ticks, i.e. after set_ticks() or using a FixedLocator.
  for batch in batches:

Show Total Whole Cell Counts¶

For each batch, cell line, replicate and markerTotal number of tiles

In [27]:
to_heatmap = total_sum.rename(columns={'site_whole_cells_counts_sum':'index'})
plot_filtering_heatmap(to_heatmap, extra_index='marker', vmin=None, vmax=None,
                      xlabel = 'Total number of whole cells', show_sum=True, figsize=(6,8))

/home/labs/hornsteinlab/Collaboration/MOmaps_Noam/MOmaps/tools/preprocessing_tools/qc_utils.py:397: UserWarning: set_ticklabels() should only be used with a fixed number of ticks, i.e. after set_ticks() or using a FixedLocator.
  for batch in batches:
/home/labs/hornsteinlab/Collaboration/MOmaps_Noam/MOmaps/tools/preprocessing_tools/qc_utils.py:397: UserWarning: set_ticklabels() should only be used with a fixed number of ticks, i.e. after set_ticks() or using a FixedLocator.
  for batch in batches:

/home/labs/hornsteinlab/Collaboration/MOmaps_Noam/MOmaps/tools/preprocessing_tools/qc_utils.py:397: UserWarning: set_ticklabels() should only be used with a fixed number of ticks, i.e. after set_ticks() or using a FixedLocator.
  for batch in batches:
/home/labs/hornsteinlab/Collaboration/MOmaps_Noam/MOmaps/tools/preprocessing_tools/qc_utils.py:397: UserWarning: set_ticklabels() should only be used with a fixed number of ticks, i.e. after set_ticks() or using a FixedLocator.
  for batch in batches:

/home/labs/hornsteinlab/Collaboration/MOmaps_Noam/MOmaps/tools/preprocessing_tools/qc_utils.py:397: UserWarning: set_ticklabels() should only be used with a fixed number of ticks, i.e. after set_ticks() or using a FixedLocator.
  for batch in batches:
/home/labs/hornsteinlab/Collaboration/MOmaps_Noam/MOmaps/tools/preprocessing_tools/qc_utils.py:397: UserWarning: set_ticklabels() should only be used with a fixed number of ticks, i.e. after set_ticks() or using a FixedLocator.
  for batch in batches:

Show Cell Count Statistics per Batch¶

In [28]:
df_no_empty_sites = df_dapi[df_dapi.n_valid_tiles !=0]
plot_cell_count(df_no_empty_sites, dnls_lines_order, dnls_custom_palette, y='site_cell_count_sum', 
                title='Cell Count Average per Site (from tiles)')

plot_cell_count(df_no_empty_sites, dnls_lines_order, dnls_custom_palette, y='site_whole_cells_counts_sum',
                title='Whole Cell Count Average per Site')

plot_cell_count(df_no_empty_sites, dnls_lines_order, dnls_custom_palette, y='site_cell_count',
               title='Cellpose Cell Count Average per Site')

Show Tiles per Site Statistics¶

In [29]:
df_dapi.groupby(['cell_line_cond']).n_valid_tiles.mean()
Out[29]:
cell_line_cond
TDP43 Untreated    6.463026
TDP43 dox          7.486036
WT Untreated       3.277463
Name: n_valid_tiles, dtype: float64
In [6]:
df_dapi[['site_cell_count']].mean()
Out[6]:
site_cell_count    16.276892
dtype: float64
In [30]:
plot_catplot(df_dapi, custom_palette,reps, x='n_valid_tiles', x_title='valid tiles count', batch_min=2, batch_max=5)

/home/labs/hornsteinlab/Collaboration/MOmaps_Noam/MOmaps/tools/preprocessing_tools/qc_utils.py:1073: SettingWithCopyWarning: 
A value is trying to be set on a copy of a slice from a DataFrame.
Try using .loc[row_indexer,col_indexer] = value instead

See the caveats in the documentation: https://pandas.pydata.org/pandas-docs/stable/user_guide/indexing.html#returning-a-view-versus-a-copy
  hue_order=palette.keys()

Show Mean of cell count in valid tiles¶

In [31]:
plot_hm(df_dapi, split_by='rep', rows='cell_line_cond', columns='panel', figsize=(10,3))

Assessing Staining Reproducibility and Outliers¶

In [8]:
for batch in batches:
    print(batch)
    run_calc_hist_new(f'{batch}', dnls_cell_lines_for_disp, dnls_markers,
                      root_directory_raw, root_directory_proc,
                           hist_sample=10,sample_size_per_markers=200, ncols=8, nrows=4, dnls=True)
    print("="*30)

batch3

==============================
batch4

==============================
batch5

==============================
In [32]:
# save notebook as HTML ( the HTML will be saved in the same folder the original script is)
from IPython.display import display, Javascript
display(Javascript('IPython.notebook.save_checkpoint();'))
os.system(f'jupyter nbconvert --to html tools/preprocessing_tools/qc_reports/qc_report_dNLS.ipynb --output {NOVA_HOME}/manuscript/preprocessing_qc_reports/qc_report_dNLS.html')

[NbConvertApp] Converting notebook tools/preprocessing_tools/qc_reports/qc_report_dNLS.ipynb to html
[NbConvertApp] Writing 13039835 bytes to /home/labs/hornsteinlab/Collaboration/MOmaps_Noam/MOmaps/manuscript/preprocessing_qc_reports/qc_report_dNLS.html
Out[32]:
0
In [ ]: